OBJECTIVE: 5-Azacytidine is a hypomethylating agent that is used for the treatment of myelodysplastic syndrome. This histone modifier is widely employed and plays a nonspecific role in influencing the differentiation capability of stem cells. The ability of bone marrow mesenchymal stem cells to differentiate into cardiomyocyte- and myocyte-like cells after exposure to 3 different doses of 5-azacytidine has been evaluated and compared. The aim of the study was to optimize the effective dose of 5-azacytidine for promoting the cardiomyocyte and myocyte differentiation capabilities of human mesenchymal stem cells (MSCs).METHODS: Human bone marrow aspirations were collected from healthy donors. MSCs were used for the study of mesodermal differentiation. MSCs were cultured to promote osteoblast differentiation and adipocyte differentiation. The evaluation of osteogenic or adipogenic properties was then performed through immunocytochemical staining. BMMSCs were trypsinized into single-cell suspensions and then prepared for flow cytometric analysis. The MSCs were treated with 5, 10, or 15 μM 5-azacytidine for 24 h and then cultured for 3 weeks. Total RNA was extracted from untreated and 5-azacytidinetreated cells. Troponin T and GATA4 antibodies were used as cardiogenic markers, whereas myogenin and MyoD antibodies were used as myocyte markers. RESULTS: The morphology and growth rate of MSCs that were treated with any of the 3 doses of 5-azacytidine were similar to the morphology and growth rate of control MSCs. An immunofl uorescence analysis examining the expression of the cardiac-specifi c markers GATA4 and troponin T and the skeletal muscle-specifi c markers MyoD and myogenin revealed that cells treated with 15 μM 5-azacytidine were strongly positive for these markers. Real-time RT-PCR results were examined; these amplifi cations indicated that there were higher expression levels of cardiac- and skeletal muscle-specifi c mRNAs in MSCs treated with 15 μm 5-azacytidine than in MSCs that had either been treated with lower doses of 5-azacytidine or left untreated.CONCLUSION: MSCs treated with 5-azacytidine demonstrated the capacity to differentiate into both cardiomyocytes and skeletal myocytes, and 15 μM 5-azacytidine could be the optimal dose of this drug. Other promoting factors should be examined to investigate the possibility of promoting the differentiation of MSCs into specific cell types.